Transcript Light, Matter, and the basics - Purdue University Cytometry

BMS 631 - LECTURE 3
Light and Matter
J.Paul Robinson
Professor of Immunopharmacology
School of Veterinary Medicine, Purdue University
Hansen Hall, B050
Purdue University
Office: 494 0757
Fax 494 0517
email\; [email protected]
WEB http://www.cyto.purdue.edu
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Shapiro p 75-93
Light and Matter
• Energy
– joules, radiant flux (energy/unit time)
– watts (1 watt=1 joule/second)
• Angles
– steradians - sphere radius r - circumference is
2r2; the angle that intercepts an arc r along
the circumference is defined as 1 radian. (57.3
degrees) a sphere of radius r has a surface area
of 4r2. One steradian is defined as the solid
angle which intercepts as area equal; to r2 on the
sphere surface
Shapiro p 75
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Terms
•
•
Side scatter, forward angle scatter, cell volume, coulter volume:
Understand light scattering concepts; intrinsic and extrinsic parameters
•
•
Photometry:
Light - what is it - wavelengths we can see 400-750 nm, most sensitive around 550 nm.
Below 400 nm essentially measuring radiant energy. Joules (energy) radiant flux
(energy per unit time) is measured in watts (1 watt=1 joule/second).
Steradian (sphere radius r has surface area of 4 r2; one steradian is defined as that
solid angle which intercepts an area equal to r2 on the surface.
Mole - contains Avogadro's number of molecules (6.02 x 1023) and contains a mass in
grams = molecular weight. Photons - light particles - waves - Photons are particles
which have no rest mass - pure electromagnetic energy - these are absorbed and
emitted by atoms and molecules as they gain or release energy. This process is
quantized, is a discrete process involving photons of the same energy for a given
molecule or atom. The sum total of this energy gain or loss is electromagnetic
radiation propagating at the speed of light (3 x 108 m/s). The energy (joules) of a
photon is
E=hn and E=hn/l [n-frequency, l-wavelength, h-Planck's constant 6.63 x 10-34 jouleseconds]
Energy - higher at short wavelengths - lower at longer wavelengths.
•
•
•
•
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Photons and Quantum Theory
• Photons
– particles have no rest mass - composed of pure
electromagnetic energy - the absorption and emission of
photons by atoms and molecules is the only mechanism for
atoms and molecules can gain or lose energy
• Quantum mechanics
– absorption and emission are quantized - i.e. discrete process
of gaining or losing energy in strict units of energy - i.e.
photons of the same energy (multiple units are referred to
 = wavelength
as electromagnetic radiation)
• Energy of a photon
h = Planck’s constant
(6.63 x 10-34 joule-seconds
– can be computed from its frequency (n) c = speed of light (3x108 m/s)
in hertz (Hz) or its wavelength (l) in meters from
E=hn and E=hc/
Shapiro p 76
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Light – Particles and waves - Reflection
Diagrams from: http://micro.magnet.fsu.edu/primer/java/particleorwave/reflection/index.html
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Light – Particles and waves -
Refraction
Diffraction
Diagrams from: http://micro.magnet.fsu.edu/primer/java/particleorwave/reflection/index.html
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Laser power
E=hn and E=hc/
• One photon from a 488 nm argon laser has an
energy of
E=
6.63x10-34 joule-seconds x 3x108
488 x 10-3
= 4.08x10-19 J
• To get 1 joule out of a 488 nm laser you need
2.45 x 1018 photons
• 1 watt (W) = 1 joule/second a 10 mW laser at
488 nm is putting out 2.45x1016 photons/sec
Shapiro p 77
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
What about a UV laser?
E=
6.63x10-34 joule-seconds x 3x108
325 x 10-3
= 6.12 x 10-19 J so 1 Joule at 325 nm = 1.63x1018 photons
What about a He-Ne laser?
E=
6.63x10-34 joule-seconds x 3x108
633 x 10-3
= 3.14 x 10-19 J so 1 Joule at 633 nm = 3.18x1018 photons
Shapiro p 77
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Polarization and Phase:
Interference
• Electric and magnetic fields
are vectors - i.e. they have
both magnitude and
direction
• The inverse of the period
(wavelength) is the
frequency in Hz
Shapiro p 78
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Interference
0o 90o 180o 270o 360o
Wavelength
Amplitude
A+B
A
Constructive
Interference
B
C+D
C
D
The frequency does
not change, but the
amplitude is doubled
Here we have a phase difference of
180o (2 radians) so the waves
cancel each other out
Destructive
Interference
Figure modified from Shapiro “Practical Flow
Cytometry” Wiley-Liss, p79
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Light Scatter
• Materials scatter light at wavelengths at which they do
not absorb
• If we consider the visible spectrum to be 350-850 nm
then small particles (< 1/10 ) scatter rather than absorb
light
• For small particles (molecular up to sub micron) the
Rayleigh scatter intensity at 0o and 180o are about the
same
• For larger particles (i.e. size from 1/4 to tens of
wavelengths) larger amounts of scatter occur in the
forward not the side scatter direction - this is called Mie
Scatter (after Gustav Mie) - this is how we come up with
forward scatter be related to size
Shapiro p 79
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Rayleigh Scatter
• Molecules and very small particles do
not absorb, but scatter light in the
visible region (same freq as
excitation)
• Rayleigh scattering is directly
proportional to the electric dipole
and inversely proportional to the 4th
power of the wavelength of the
incident light
the sky looks blue because the gas molecules scatter more
light at shorter (blue) rather than longer wavelengths (red)
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Reflection and Refraction
Incident Beam
i
r
Reflected Beam
• Snell’s Law: The angle of
reflection (Ør) is equal to
Transmitted
the angle of incidence
(refracted)Beam
(Øi) regardless of the
surface material
t
• The angle of the
transmitted beam (Øt) is
dependent upon the
composition of the
material
n1 sin Øi = n2 sin Øt
The velocity of light in a material
of refractive index n is c/n
Shapiro p 81
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Refraction & Dispersion
rac
Short wavelengths are “bent”
more than long wavelengths
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Brewster’s Angle
• Brewster’s angle is the angle at which the reflected
light is linearly polarized normal to the plane incidence
• At the end of the plasma tube, light can leave through
a particular angle (Brewster’s angle) and essentially be
highly polarized
• Maximum polarization occurs when the angle between
reflected and transmitted light is 90o
thus Ør + Øt = 90o
since sin (90-x) = cos x
Snell’s provides (sin Øi / cos Øi ) = n2/n1
Ør = tan -1 (n2/n1)
Ør is Brewster’s angle
Shapiro p 82
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Brewster’s Angle
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Interference in Thin Films
• Small amounts of incident light are reflected at
the interface between two material of different
RI
• Thickness of the material will alter the
constructive or destructive interference patterns
- increasing or decreasing certain wavelengths
• Optical filters can thus be created that
“interfere” with the normal transmission of light
Shapiro p 82
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Interference and Diffraction:
Gratings
• Diffraction essentially describes a
departure from theoretical geometric
optics
• Thus a sharp objet casts an alternating
shadow of light and dark “patterns”
because of interference
• Diffraction is the component that
limits resolution
Thomas Young’s double split
experiment in 1801
http://micro.magnet.fsu.edu/primer/java/interference/doubleslit/index.html
Shapiro p 83
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Absorption
• Basic quantum mechanics requires that molecules
absorb energy as quanta (photons) based upon a
criteria specific for each molecular structure
• Absorption of a photon raises the molecule from
ground state to an excited state
• Total energy is the sum of all components
(electronic, vibrational, rotational, translations,
spin orientation energies) (vibrational energies are
quite small)
• The structure of the molecule dictates the likelyhood of absorption of energy to raise the energy
state to an excited one
Shapiro p 84
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence Lifetime
•
•
•
•
•
Absorption associated with electronic transitions (electrons changing states) occurs
in about 1 femptosecond (10-15 s)
Fluorescence lifetime is defined as the time in which the initial fluorescence
intensity of a fluorophore decays to 1/e (approx 37 percent) of the initial intensity
The lifetime of a molecule depends on how the molecule disposes of the extra energy
Because of the uncertainty principle, the more rapidly the energy is changing, the
less precisely we can define the energy
So, long-lifetime-excited-states have narrow absorption peaks, and short-lifetimeexcited-states have broad absorption peaks
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Shapiro p 85
Exctinction
• Using Beer’s law (Beer-Lambert law) for light travelling through
a curvette thickness d cm containing n molecules/cm3
ln (Io/I) = nd
where Io and I are the light entering and leaving and  is the molecular
property called the absorption cross section
• Now we can state that
ln (Io/I) = nd where C is the concentration and a is the
absorption coefficient which reflects the capacity of the absorbing
substance to absorb light
• If there are n (molecules/cm3 ; d in cm,  must be in cm2 so if 
is in cm2/mol, C must be in mol/cm3 do C=a/103
• giving
log10 (Io/I) = d = A
where A is the absorbance or optical density
and  is the decadic molar exctinction coeficient in dm3mol-1cm-1
Shapiro p 86
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Absorbance
• O.D. units or absorbance is expressed in logarithmic
terms so they are additive.
• E.g. an object of O.D. of 1.0 absorbs 90% of the
light. Another object of O.D. 1.0 placed in the path
of the 10% of the light 10% of this light or 1% of
the original light is transmitted by the second object
• It is posssible to express the absorbance of a
mixture of substances at a particular wavelength as
the sum of the absorbances of the components
• You can calculate the cross sectional area of a
molecule to determine how efficient it will absorb
photons. The extinction coefficient indicates this
value
Shapiro p 87
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
• Photon emission as an electron
returns from an excited state to
ground state
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Parameters
• Extinction Coefficient
– 
refers to a single wavelength (usually the absorption maximum) (The
extinction coefficient is determined by measuring the absorbance at a
reference wavelength (characteristic of the absorbing molecule) for a one
molar (M) concentration (one mole per liter) of the target chemical in a cuvette
having a one-centimeter path length.)
– the intrinsic lifetime of a fluorophore is inversely proportional to the extinction
coefficient, molecules exhibiting a high extinction coefficient have an excited
state with a short intrinsic lifetime.
• Quantum Yield
– Qf
is a measure of the integrated photon emission over the fluorophore
spectral band
– Expressed as ratio of photons emitted to the number of photons absorbed
(zero to 1 (best)
• At sub-saturation excitation rates, fluorescence
intensity is proportional to the product of  and Qf
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
• Quantum Yield
Q=
photons emitted
=
photons absorbed
kr
kr + knr
• Fluorescence Lifetime ()
- is the time delay between the absorbance and
the emission

1
= k +k
r
nr
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
• Excitation Spectrum
– Intensity of emission as a function of
exciting wavelength
• Chromophores are components of
molecules which absorb light
• They are generally aromatic rings
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
• The wavelength of absorption is
related to the size of the
chromophores
• Smaller chromophores, higher energy
(shorter wavelength)
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
• Stokes Shift
Fluorescnece Intensity
– is the energy difference between the
lowest energy peak of absorbance and
the highest energy of emission
Fluorescein
molecule
Stokes Shift is 25 nm
495 nm
520 nm
Wavelength
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
• The longer the wavelength the lower the
energy
• The shorter the wavelength the higher the
energy
– eg. UV light from sun - this causes the sunburn,
not the red visible light
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Electromagnetic Spectrum
© Microsoft Corp, 1995
Only a very small region within the ES
is used for flow cytometry applications
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Properties of Fluorescent
Molecules
Large extinction coefficient at the
region of excitation
 High quantum yield
 Optimal excitation wavelength
 Photostability
 Excited-state lifetime
 Minimal perturbation by probe

© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Simplified Jablonski Diagram
S’
1
S1
hvex
hvem
S0
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
Jablonski Diagram
Singlet States
Triplet States
Vibrational energy levels
Rotational energy levels
Electronic energy levels
S2
ENERGY
T2
S1
IsC
T1
ABS
FL
I.C.
PH
IsC
S0
[Vibrational sublevels]
ABS - Absorbance
S 0.1.2 - Singlet Electronic Energy Levels
FL - Fluorescence
T 1,2 - Corresponding Triplet States
I.C.- Nonradiative Internal Conversion IsC
- Intersystem Crossing
PH - Phosphorescence
Shapiro p 87
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
The longer the wavelength the lower the energy
The shorter the wavelength the higher the energy
eg. UV light from sun causes the sunburn
not the red visible light
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence Excitation
Spectra
Intensity
related to the probability of the event
Wavelength
the energy of the light absorbed or emitted
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Some Conclusions
• Dye molecules must be close to but below
saturation levels for optimum emission
• Fluorescence emission is longer than the
exciting wavelength
• The energy of the light increases with
reduction of wavelength
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
350
300 nm
457 488 514
400 nm
500 nm
Common Laser Lines
610 632
600 nm
700 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Allophycocyanin (APC)
Protein
300 nm
400 nm
500 nm
632.5 nm (HeNe)
600 nm
700 nm
Excitation
Emisson
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorochrome excitation and emission
spectra
Typical fluorchromes
FITC
PE
PerCP-Cy5.5
PE-Cy7
http://m.bdbiosciences.com/us/s/spectrumviewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
The problem of spectral overlap
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Excitation Saturation
• The rate of emission is dependent upon the time the molecule
remains within the excitation state (the excited state lifetime
f)
• Optical saturation occurs when the rate of excitation exceeds
the reciprocal of f
• In a scanned image of 512 x 768 pixels (400,000 pixels) if
scanned in 1 second requires a dwell time per pixel of 2 x 10-6
sec.
• Molecules that remain in the excitation beam for extended
periods have higher probability of interstate crossings and thus
phosphorescence
• Usually, increasing dye concentration can be the most effective
means of increasing signal when energy is not the limiting factor
(i.e. laser based confocal systems)
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Phosphorescence
• Following absorption, molecules can relax via a nonradiative transition to the T1 rather than the S1 state this is called an intersystem crossing,
• While it is forbidden it does happen and has a low
probability and takes a longer time - the energy
dissipated is called phosphorescence
• Phosphorescence has a longer lifetime than
fluorescence (milliseconds rather than femptoseconds
• Phosphorescence generally occurs at longer
wavelengths than fluorescence because the energy
difference between S0 and T1 is lower
Shapiro p 88
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Resonance Energy Transfer
• Resonance energy transfer can occur when the
donor and acceptor molecules are less than 100 A
of one another
• Energy transfer is non-radiative which means the
donor is not emitting a photon which is absorbed
by the acceptor
• Fluorescence RET (FRET) can be used to
spectrally shift the fluorescence emission of a
molecular combination.
Shapiro p 90
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Fluorescence
Resonance Energy
Transfer
Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Absorbance
Absorbance
Wavelength
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Tandem conjugates
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
APC is exited nicely by 632 nm
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
APC and the Tandem
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
And now with 3 probes from 632 nm
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Change Excitation – nothing!
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
PE-tandems
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
PE Tandems with 561 Excitation – more efficient
From BD Spectra viewer
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Raman Scatter
• A molecule may undergo a vibrational transition (not
an electronic shift) at exactly the same time as
scattering occurs
• This results in a photon emission of a photon
differing in energy from the energy of the incident
photon by the amount of the above energy - this is
Raman scattering.
• The dominant effect in flow cytometry is the
stretch of the O-H bonds of water. At 488 nm
excitation this would give emission at 575-595 nm
Shapiro p 93
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Quenching, Bleaching &
Saturation
• Quenching is when excited molecules relax to
ground stat5es via nonradiative pathways avoiding
fluorescence emission (vibration, collision,
intersystem crossing)
• Molecular oxygen quenches by increasing the
probability of intersystem crossing
• Polar solvents such as water generally quench
fluorescence by orienting around the exited state
dipoles
Shapiro p 90
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt
Summary
• Review of nature of light
• Review of fundamental features of
light
• Review of fluorescence properties
• Review of how de define efficiency of
light
• Review of fluorescence
• Review of factors that influence light
(quenching, lifetime, etc)
© 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt